Purification of hop extracts

ABSTRACT

Xanthohumol is removed from a hop extract solution by treatment with a polyamide, for example by passing the solution down a chromatographic column of polyamide or by slurrying together and separating the insoluble polyamide. Polyvinyl pyrrolidone is preferred. The treated extract can be used in brewing with or without further treatment.

United States Patent [191 Gardner [451 Feb. 26, 1974 PURIFICATION OF HOPEXTRACTS [75] Inventor: David Stanley John Gardner,

Reigate, England [73] Assigneez- White Tomkins Limited,, Reigate,Surrey, England [22] Filed: May 10, 1972 [21] Appl. No.: 252,331

[52] US. Cl. 426/424 [51] Int. Cl ..A23l,C12h I100 [58] Field of Search99/505, 5], 52

[56] References Cited OTHER PUBLICATIONS Ashurst et al. Chemistry of HopConstituents XXV Xanthohumol Content J. Institute of Brewing, Vol. 71,pp. 492-495, (1965).

PrimaryExaminer-Li0nel M. Shapiro Assistant Examiner-R. B. Penland [57]ABSTRACT 7 Claims, N0 Drawings PURIFICATION OF HOP EXTRACTS BACKGROUNDOF THE INVENTION i This invention relates to the purification of hopextracts and particularly to the removal of xanthohumol and analogousmaterials from hop extracts.

The main component of the hard resin fraction of -hop resins is thechalkone xanthohumol. This material has been shown by Ashurst, Laws andStevens (.1. Inst. Brew. US. Pat. No. 71,492 (1965) to be present inamount of almost 1 percent of the dry weight of fresh or unkilned,freeze-dried hops and to be present in an amount of about 0.3 percent inoast dried sulphured hops. Other analogous chalkones may also be presentand the term xanthohumol as used herein includes such analogousmaterials. Most solvents which are used to prepare hop extracts willextract some xanthohumol, the amount extracted depending on the polarityof the solvent used. When hops, or solvent extracts of hops, containingxanthohumol are employed in the traditional brewing process (whichinvolves addition of the hops, or extracts, to the wort prior toboiling) the water insoluble portion of the hard resins, including thexanthohumol is rejected and removed. However, some unwantedco-precipitation of the desirable humulones with the xanthohumol occurs.Furthermore, in recent years it has been found advantageous to add hopextracts to the beer after completion of fermentation, the extractshaving been first isomerised to convert the humulones present to thedesirable bitter compounds the isohumulones. Such processes aredescribed, for example, in U.K. Patent Specification No. 1,259,140.Unless steps have been taken to remove xanthohumol, some of thismaterial, together with the isomerised product, the flavanoneisoxanthohumol, is carried through to the final product and,particularly when added to beer, tends to cause unwanted turbidity. Thisis particularly so since xanthohumol is slightly soluble in water in theform of its alkali metal salts, but is virtually insoluble in the formof free acid.

SUMMARY OF THE INVENTION It is an object of this invention to overcomethis problem by means of a method of removing xanthohumol from a hopextract comprising contacting the hop extract in the form of a solutionin a suitable solvent with a polyamide.

DESCRIPTION OF PREFERRED EMBODIMENTS The preferred polyamide ispolyvinylpyrrolidone. A convenient solvent is methanol, but otherappropriate solvents, such as other alcohols, esters, ketones orhydrocarbons may be used.

The hop extract may contain both hard and soft resins in which case, ifthe solvent used is methanol, the xanthohumol freed methanolic solutionafter its removal from the polymide, can be extracted with petroleumspirit to extract the soft resin fraction of the hop extract without theneed for an intermediate isolation step.

Such removal of xanthohumol before the petroleum spirit extraction isuseful as, if xanthohumol is still present, the extraction tends to beincomplete and the continued presence of some xanthohumol can lead tothe entraining of some of the desirable humulones in the interfacialmaterial.

However, the method for removing xanthohumol can also be applied to hopextracts from which other hard resins have been at leats partiallyremoved or the method can even be applied to a xanthohumolcontaininghard resin fraction itself to remove the xanthohumol selectively.

The polyamide material may be used in the form of a chromatographiccolumn through which the hop extract solution is passed and thexanthohumol can later be eluted, using for example water-saturated ethylacetate. Alternatively, the polyamide material may simply be slurriedwith the solution. The absorbed xanthohumols may be recovered from thepolyamide by' solvent extraction. Alternatively they may be chemicallyconverted to compounds having no polyamide affinity, thus releasing theminto a suitable solvent.

After contacting with the polyamide, the solution can be evaporated toyield an extract which can be further treated as desired, used directlyin the traditional brewing process or dissolved in a water immisciblesolvent for conversion to an isomerised extract as described in U.K.Patent Specification No. 1,259,140,

The following specific examples illustrate the inventron:

EXAMPLE I 135 grms. of a hop extract estimated to contain 43 percentalpha-acids by conducto'metric titration, prepared by extracting hopswith toluene and ridding the crude product of fixed oils, waxes and fatby treatment with percent methanol, were re-dissolved in methanolpercent) and thevolume was made up to 300 mls. This solution was passeddown a chromatographic column prepared with 25 grms.Polyvinylpyrrolidone (Polyclar A.T.) 25 grms. Hyflo Supercel, wet packedwith methanol, to give a column 4 inches long and 2 inches diameter.Polyclar A.T. is available from General Aniline and Film Corp. and HyiloSupercel is one of a range of suitable filter aids available from theZeo' lite division of Johns Manville.

The column was developed with methanol until 600 mls. of eluate had beencollected. At this point virtually all the coloured material had passedthrough the column except for a small pink colouredband at the topof thecolumn together with the xanthohumol which.

was present a few cms. below this as a compact yellow band.

Evaporation of the primary eluate gave 131.5 grms. of purified resinsestimated to contain 44 percent alpha-acids.

Elution of the column with ethyl acetate (water saturated) until all thecolour had been removedfollowed by evaporation of the solvent yielded3.5 grms. crystalline xanthohumol (M.pt. 172 C). The purified resins,the starting material and the xanthohumol fractions were all examined bythin layer chromatography for the presence of xanthohumol and other hopconstituents by the procedure described below. No xanthohumol wasdetected in the purified resin.

THIN LAYER CHROMATOGRAPHY PROCEDURE Plater with a 0.25 mm. layer ofsilica gel (Merck Kieselgel G.) were activated by heating at C for 30minutes and then contracted with the solution to be examined. Thedeveloping solvent used was ethyl acetate: benzene 1:1. The sprayreagent used was 2 percent ferric chloride in pure methanol or percentformalin in methanol containing 1 percent sulphuric acid. On heating theplates, xanthohumol if present, appeared as a spot, well separated fromthe alpha-and beta-acids (if present), appearing approximately mid waybetween them. The spot was yellow ochre in colour with ferric chloridereagent and cherry red with the formalin.

EXAMPLE 2 3,000 grms. of a de-solventised extract of OR55 Hops preparedwith toluene and estimated to contain 36.2 percent alpha-acids werewarmed and poured into 2 gallons of 100 percent methanol, decanted fromthe methanol insoluble waxes and fats (51 2 grms. insolubles) which werethen washed by resuspending in fresh methanol and allowing to settleonce again. The bulked primary methanol extract and washings werestirred for 30 minutes with 500 grms. of polyvinylpyrrolidone in theinsoluble form (Polyclar A.T.). The methanol solution was decanted fromthe Polyclar, which was washed with clean methanol, the washings beingadded to the bulk. The methanol was distilled off, finally under vacuum,to give a resin free of xanthohumol.

Yield 2,161 grms. Estimated 48.2 percent alphaacids by titration.

Recovery of alpha-acids 95.8 percent.

EXAMPLE 3 387 grms. hard resin fraction derived from a hop extractcontaining 12.5 percent of xanthohumol (determined from the ion-exchangechromatogram by calculating the area under the curve) were dissolved inpure methanol and the solution was chromatographed on a column ofpolyvinyl-pyrrolidone. (250 grms. Polyclar A.T. 250 grms. Hyflo Supercelin 4 inch diameter by 24 inch column.)

The column was developed with methanol until the frontal band materialhad been eluted, leaving the yellow xanthohumol zone. The elutingsolvent was then changed to water equilibrated ethyl acetate and thecolumn was washed with this until all yellow colour had been eluted.

Evaporation of the methanol yielded 317 grms. of xanthohumol free resinand evaporation of the ethyl acetate under a partial vacuum yielded 51grms. of xan thohumol which immediately crystallised upon removal of thelast traces of solvent. This yield of relatively pure xanthohumol is inagreement with that determined by ion-exchange chromatography.

EXAMPLE 4 fats amounted to 217 g.

The primary methanol extract (a) was applied to the top of a column (6inches diameter X 12 inches long) containing a mixture of 750 g.Polyclar AT 750 g. Hyflo Supercel. The column was developed with themethanol extracts (b) and (c) and finally washed with fresh methanol.The xanthohumol was clearly visible as an intense yellow band occupyingabout three-fourths of the column length. The xanthohumol was completelyremoved by elution with ethyl acetate-saturated with water and thecolumn washed, ready for re-use, with pure methanol. Evaporation of theethyl acetate eluate yielded crystalline xanthohumol (124 grms.).

The methanol eluate was adjusted with water to percent methanol byvolume and extracted with light petroleum (10 litres) followed by twofurther extractions (5 litres each) which were all bulked. Evaporationof the petrol extracted aqueous methanol solution gave a xanthohumolfree hard resin (203 grrns). The bulked petrol extracts yielded onevaporation the desired soft resin fraction (2,456 grms.) containing50.0 percent by weight of alpha-acids.

I claim:

1. In a method-of removing xanthohumol from a hop extract dissolved in asuitable solvent, the improvement consisting of the steps of contactingthe solution of the hop extract with polyvinylpyrrolidone and separatingthe solution from the polyvinyl pyrrolidone whereby the xantholhumol isseparated from the solution by remaining with the polyvinyl pyrrolidone.

2. The improvement as claimed in claim 1 wherein the solvent ismethanol.

3. A method of removing xanthohumol from a hop extract which comprisesthe steps of making a solution of a hop extract in a suitable solvent,contacting the solution with a polyvinyl pyrrolidone in insoluble formto sequester the xanthohumol and separating the solution from thepolyvinyl pyrrolidone.

4. A method of separating soft resins and hard resins in a hop extractcontaining soft and hard resins which comprises the steps of making asolution of the hop extract in methanol, contacting the solution with apolyvinyl pyrrolidone in insoluble form to remove xanthohumol from thesolution, separating the solution from the polyvinyl pyrrolidone, andextracting the solution with petroleum spirit to separate the softresins from the remaining hard resins.

5. A method according to claim 1 wherein the hop extract solution iscontacted with the polyvinyl pyrrolidone by passing the solution througha chromatographic column of the polyvinyl pyrrolidone in insoluble form.

6. A method according to claim 5 including the further steps ofsubsequently eluting the removed xanthohumol from the chromatographiccolumn with water-saturated ethyl acetate.

7. A method according to claim 1 wherein the hop extract solution iscontacted with the polyvinyl pyrrolidone by slurrying the solution withthe polyvinyl pyrrolidone in insoluble form.

2. The improvement as claimed in claim 1 wherein the solvent ismethanol.
 3. A method of removing xaNthohumol from a hop extract whichcomprises the steps of making a solution of a hop extract in a suitablesolvent, contacting the solution with a polyvinyl pyrrolidone ininsoluble form to sequester the xanthohumol and separating the solutionfrom the polyvinyl pyrrolidone.
 4. A method of separating soft resinsand hard resins in a hop extract containing soft and hard resins whichcomprises the steps of making a solution of the hop extract in methanol,contacting the solution with a polyvinyl pyrrolidone in insoluble formto remove xanthohumol from the solution, separating the solution fromthe polyvinyl pyrrolidone, and extracting the solution with petroleumspirit to separate the soft resins from the remaining hard resins.
 5. Amethod according to claim 1 wherein the hop extract solution iscontacted with the polyvinyl pyrrolidone by passing the solution througha chromatographic column of the polyvinyl pyrrolidone in insoluble form.6. A method according to claim 5 including the further steps ofsubsequently eluting the removed xanthohumol from the chromatographiccolumn with water-saturated ethyl acetate.
 7. A method according toclaim 1 wherein the hop extract solution is contacted with the polyvinylpyrrolidone by slurrying the solution with the polyvinyl pyrrolidone ininsoluble form.